Composition for the prevention and treatment of kidney dysfunctions and diseases

ABSTRACT

A composition is disclosed which is suitable for the prevention and treatment of kidney dysfunctions and diseases and which can therefore take the form of a food supplement or of an actual medicinal drug comprising a combination of acetyl L-carnitine and propionyl L-carnitine or their pharmacologically acceptable salts.

This application is a divisional of application Ser. No. 10/019,545,filed Jan. 3, 2002, now U.S. Pat. No. 6,653,349, which in turn is a 35U.S.C. 371 of PCT/IT00/00313 filed Jul. 25, 2000.

The present invention relates to a composition suitable for theprevention and treatment of kidney dysfunctions and diseases.

Correspondingly, the composition may take the form and exert theactivity of a food supplement or of an actual medicinal drug, dependingupon the support or preventive action or the strictly therapeutic actionwhich the composition is intended to exert according to the particularindividuals for whom it is to be used.

More specifically, the composition according to the present invention issuitable for the prevention and treatment of all forms of nephropathy,whether caused by external agents such as nephrotoxic drugs likelithium, antibiotics and anticancer drugs with a nephrotoxic potential,environmental contaminants such as mycotoxins of the ochratoxin type, ordue to kidney function deficits of metabolic origin.

The composition according to the present invention comprises as itscharacterising ingredients acetyl L-carnitine and propionyl L-carnitineor the pharmacologically acceptable salts thereof which will beidentified hereinbelow.

The use of “carnitines” in the field of nephrology (where the term“carnitines” refers collectively to both L-carnitine and to the loweralkanoyl L-carnitines such as acetyl, propionyl butyryl L-carnitine,etc.) is already known.

U.S. Pat. Nos. 4,272,549 and 4,237,167 (Sigma-Tau IndustrieFarmaceutiche Riunite S.p.A) describe the use of L-carnitine and of theabove-mentioned respective alkanoyl L-carnitines to prepare polysalinesolutions which are fed into the dialysis compartment of artificialkidneys. The purpose of the presence of said carnitines in the dialysisfluid is to counteract the loss of L-carnitine in the blood and tissueswhich habitually occurs in patients undergoing regular haemodialysistreatment, with the consequent onset of disorders of the skeletal muscleand myocardium.

It is equally well known that a lack of L-carnitine may be the cause oftubular necrosis following kidney transplantation.

Finally, EP 0722 724 discloses the use of L-carnitine or lower alkanoylL-carnitine for preparing a medicament apt to inhibit nephro- andvasculotoxicity induced by the administration of immunosuppressants suchas cyclosporin-A, tacrolimus, rapamicine and deoxyspargualine; and DrugsExptl. Clin Res. XXI (6), 221-228 (1995) specifically reports on theprotective effect of propionyl L-carnitine on cyclosporine-inducednephrotoxicity.

In contrast, neither known nor suggested by the vast amount of knowledgeavailable on the role of the carnitines and their possible therapeuticapplications, particularly as developed over the past few decades, isthe act that the composition according to the present invention,consisting of a combination of acetyl L-carnitine and propionylL-carnitine or the pharmacologically acceptable salts thereof presents apotent kidney-protecting action, particularly as regards the preventionand treatment of kidney function impairments caused by toxic agents ormetabolism disorders.

In the combination composition according to the invention the w/w ratioof acetyl L-carnitine to propionyl L-carnitine ranges from 1:1 to 1:10.

The composition may also contain an additional carnitine selected fromthe group consisting of L-carnitine, butyryl L-carnitine, valerylL-carnitine and isovaleryl L-carnitine or their pharmacologicallyacceptable salts. The w/w ratio of acetyl L-carnitine and propionylL-carnitine to this additional carnitine ranges from 1:0.5 to 1:2.

Additional optional components comprise vitamins, coenzymes, mineralsand antioxidants.

It is advisable to administer 2-5 mg of acetyl L-carnitine and 2-5 mg ofpropionyl L-carnitine per kg body weight per day, though higher dosescan also be administered in view of the very low toxicity of thecomponents.

The marked kidney-protecting activity exerted by the combination ofacetyl L-carnitine and propionyl L-carnitine is shown by the results ofa number of experimental tests which are reported here below.

These tests were selected in such a way as to be predictive of theefficacy of the combination in human subjects and in the clinical field.They also demonstrate the surprisingly potent synergistic action exertedwhen acetyl L-carnitine and propionyl L-carnitine are used incombination.

TOXICOLOGY TESTS

Both acetyl L-carnitine and propionyl L-carnitine are known to becharacterised by very low toxicity. Toxicology tests showed that evenhigh doses of acetyl L-carnitine (750 mg/kg) in combination withpropionyl L-carnitine (750 mg/kg) administered intraperitoneally to ratsdid not cause the deaths of any of the animals thus treated. The sameresult was obtained with the oral administration of 1.25 g of acetylL-carnitine together with 1.25 g of propionyl L-carnitine.

The oral administration of 500 mg/kg of acetyl L-carnitine together with500 mg/kg of propionyl L-carnitine for thirty consecutive days was alsowell tolerated.

No abnormalities of body growth or blood crasis were detected, in fact,as compared to control animals, neither were any abnormalities detectedin the animals thus treated after performing blood-chemistry tests.

Anatomico-pathological and histological examinations carried out on themain organs and tissues also failed to reveal any significantabnormalities, thus confirming the very low toxicity and goodtolerability of the two compounds used in combination.

Protective Activity Against Experimentally Induced Renal Insufficiency

To assess the kidney-protecting activity exerted by the combination ofacetyl L-carnitine and propionyl L-carnitine, renal insufficiencyresembling the so-called “crush syndrome” in man (Stein J. H., LifschitzM. D., Bernes L. D., Am. J. Physiol: 234, F171, 1978) was experimentallyinduced in rats by intramuscular injection of glycerol, as described byYoung (Young J. H. K., Meth. Find. Exptl. Clin. Pharmacol., 13, 23,1991). For this purpose, different groups of anaesthetised maleSprague-Dawley rats with a mean weight of 300 g, deprived of drinkingwater for the previous 24 hours, received intramuscular injections of 10mg/kg of an aqueous solution of 50% glycerol. Whereas the first of thesegroups was kept as a control group, the other groups were treated orallyimmediately after glycerol injection for the three days preceding thetest with 100 mg/kg or 500 mg/kg of acetyl L-carnitine, or with 100mg/kg or 500 mg/kg of propionyl L-carnitine or with the same doses ofthe two drugs in combination. Intraperitoneal administration was donefor the same period of time with 50 mg/kg or 250 mg/kg of acetylL-carnitine or with 50 mg/kg or 250 mg/kg of propionyl L-carnitine orwith the same doses of the two drugs in combination. Twenty-four hoursafter injection, blood samples were taken from the animals thus treatedas well as from the control animals. On the plasma obtained bycentrifuging, creatinine was determined according to the Taussky method(Taussky H. H., Clin. Chem. Acta, 1, 20, 1956), plasma proteinsaccording to the Lowry method (Lowry O. H., Rosembrough N. J., Fan A.L., Randall R. Y., J. Biol. Chem., 193, 265, 1951) and urea by means ofreaction with diacetyl monoxime.

The glycerol-induced acute renal insufficiency was demonstrated by asubstantial increase in plasma concentrations of both urea andcreatinine.

Administration of acetyl L-carnitine or propionyl L-carnitine aloneproduced only slight changes in the elevated plasma urea and creatinineconcentrations, whereas a surprisingly marked reduction was noted whenacetyl L-carnitine and propionyl L-carnitine were administered incombination.

The synergistic effect of acetyl L-carnitine and propionyl L-carnitinein terms of the protection afforded against glycerol-induced renalinsufficiency was marked both when acetyl L-carnitine and propionylL-carnitine were administered orally and when they were administeredintraperitoneally (see Table 1).

TABLE 1 Protective activity against glycerol-induced renal insufficiencyProteins mg/ Admin. Urea Creatinine (mg/ Treatm. kg route (mg/100 ml)(mg/100 ml) 100 ml) Con- 20.5 ± 2.1 55.8 ± 9.9 62.6 ± 5.9 trols Gly-120.5 ± 27.5 360.7 ± 25.5 66.2 ± 7.2 cerol ACL 100 oral 110.9 ± 21.6320.4 ± 20.6 65.2 ± 6.9 ACL 500 ″ 105.4 ± 29.4 315.9 ± 35.5 65.3 ± 7.2PC 100 ″ 107.4 ± 30.2 295.1 ± 28.4 65.9 ± 8.1 PC 500 ″  90.2 ± 31.4280.8 ± 30.2 64.1 ± 9.4 ACL + 100 ″ PC 100 ″  72.6 ± 12.4 165.2 ± 20.463.1 ± 6.5 ACL + 500 ″ PC 500 ″  39.7 ± 15.6  80.4 ± 14.8 62.7 ± 6.1 ACL100 i.p. 104.7 ± 12.4 300.5 ± 30.1 65.2 ± 6.6 ACL 500 ″  98.5 ± 10.6280.4 ± 21.7 64.4 ± 5.5 PC 100 ″ 100.5 ± 9.8 270.9 ± 29.4 62.4 ± 7.1 PC500 ″ 80.2 ± 6.6 260.6 ± 20.9 63.6 ± 7.5 ACL + 100 ″ PC 100 ″  55.9 ±11.5 155.1 ± 18.4 63.3 ± 5.9 ACL + 500 ″ PC 500 ″ 35.5 ± 5.9 75.2 ± 6.262.9 ± 6.3 ACL = Acetyl L-carnitine PC = Propionyl L-carnitine

Protective Activity Against Experimentally Induced Renal Toxic Lesions

In experimental animals, particularly rats, it is possible to reproducetubulo-interstitial nephropathy similar to that encountered in man andknown as Balkans Endemic Nephropathy (BEN) by means of theadministration of a mycotoxin produced by Aspergillus ochraceus which ispresent as a contaminant in various foodstuffs and is known asochratoxin A (Kniper-Goodman T., Scott P., Biomed. Environ. Sci., 2,179, 1989).

The administration of small amounts of this toxin causes a series ofrenal abnormalities in the rat with a reduction in glomerular filtrationand lesions at the level of the proximal tubule to such an extent as tocause renal atrophy. With a panel of tests conducted in rats intoxicatedwith ochratoxin A, it proved possible to demonstrate that, in thisexperimental model, too, the administration of the combination of acetylL-carnitine and propionyl L-carnitine exerts an important protectiveeffect against the renal abnormalities induced by the toxin. This effectis surprisingly greater than would be expected on summing the singleeffects obtained with the use of acetyl L-carnitine alone or propionylL-carnitine alone.

In this test a batch of Wistar rats with a mean body weight of 200 g wasdivided into various groups, one of which served as a control group,while another was administered 300 mg/kg of ochratoxin (Sigma Chemical,St. Louis, Mo. U.S.A.) by gastric probe every 48 hours for a period often days. The remaining groups, in addition to ochratoxin, also receivedoral administrations of 300 mg/ks of acetyl L-carnitine or 300 mg/kg ofpropionyl L-carnitine or a combination of the two carnitines for theentire 10-day treatment period.

At the end of treatment, the animals were placed in metabolic cages and24-hour urine was submitted to spectrophotometric evaluation of theenzymes alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT) andN-acetyl-β-D-glucosaminidase (NAG) as indicators of renal damage.

Serum creatinine concentrations were also measured.

On the basis of the results of these tests (see Table 2) the high degreeof enzymuria induced by ochratoxin and serum creatinine levels are shownto be reduced by the administration both of acetyl L-carnitine andpropionyl L-carnitine alone. However, a surprisingly greater protectiveeffect was achieved with the administration of the combination of acetylL-carnitine plus propionyl L-carnitine, thus demonstrating the potentsynergistic effect produced by the combination.

TABLE 2 Protective activity against renal abnormalities induced byochratoxin A ALP GGT NAG Serum creatinine Ochratoxin Treatm. mg/kg (U/I)(U/I) (U/I) (mg/100 ml) Cotrols 3.5 ± 0.8 11.6 ± 1.7 31.1 ± 3.3 250.6 ±19.2 Ochratoxin A 9.6 ± 1.4 18.9 ± 2.1 50.2 ± 4.6 620.4 ± 45.5 ACL 3008.8 ± 1.1 17.2 ± 1.9 48.8 ± 5.1 570.2 ± 50.4 PC 300 7.5 ± 0.9 16.5 ± 2.244.2 ± 4.5 505.9 ± 46.4 ACL + 300 PC 300 4.8 ± 1.2 13.3 ± 1.9 36.8 ± 3.9390.5 ± 30.6 ACL = Acetyl L-carnitine PC = Propionyl L-carnitineProtective Activity Against Lithium-Induced Nephrotoxicity in theIsolated Perfused Kidney

This test was used to assess the protective activity of the combinationof acetyl L-carnitine plus propionyl L-carnitine against the well-knownnephrotoxic activity of lithium. Lithium presents a particular tropismat the renal tubule level and can cause tubular necrosis.

In this test Wistar rates with a mean body weight of 400 g wereanaesthetised and nephrectomised. The excised kidneys were perfusedaccording to the technique described by Shureck (Shureck H. J., PflügersArch., 354, 1975) as modified by Maach (Maach T., Kidney Int., 30, 142,1986; Maach T., Ann. J. Physiol., 238, 1980).

Immediately after isolation of the kidney, the renal artery and outflowvein were catheterised and the resulting preparation was placed in achamber containing a Krebs solution heated to 37° C. The oxygenisedKrebs solution was pumped into the system by means of a pulsatile pumpat a rate of 0.8 ml/min/g.

Lithium was administered via the perfusion pump at a dose of 6 mEq/Lafter perfusion during the previous 20 minutes of a solution containingacetyl L-carnitine or propionyl L-carnitine (200 mM/L) or a combinationof the two compounds.

After the lithium infusion, the venous perfusion fluid was collected atfive-minute intervals for fifteen minutes and its histamine content wasmeasured with a fluorometric method (Hakanson R., Anal. Biochem., 47,356, 1972). Histamine release is considered a marker of the severity oftubular damage (Bertelli A., Drugs Exptl. Clin Res., 7, 53, 1981).

As shown by the data reported in Table 3, the histamine release inducedby the renal toxic action of lithium was reduced by the perfusion ofacetyl L-carnitine or propionyl L-carnitine, but the greatest reductionwas brought about by administration of the two carnitines incombination. The results of this test also demonstrate the potentsynergistic kidney-protecting effect exerted by the combination ofacetyl L-carnitine plus propionyl L-carnitine.

TABLE 3 Percentage changes vs controls in histamine released in theisolated kidney by lithium alone and after infusion of acetylL-carnitine or propioNyl L-carnitine or a combination of acetylL-carnitine plus propionyl L-carnitine Post-lithium-infusion time (inminutes) 5 10 15 Lithium   65 ± 5.9 42.2 ± 3.6 15.1 ± 1.9 ACL + lithium55.8 ± 6.1 36.8 ± 3.1 12.5 ± 1.5 PC + lithium 45.8 ± 4.5 30.4 ± 2.9 10.4± 0.9 ACL + PC + lithium 21.4 ± 2.5 16.2 ± 0.2  5.5 ± 0.3 ACL = AcetylL-carnitine PC = Propionyl L-carnitine

Some non-limiting examples of compositions according to the inventionare given hereinbelow.

1) Acetyl L-carnitine 1000 mg Propionyl L-carnitine 1000 mg 2) AcetylL-carnitine 500 mg Propionyl L-carnitine 500 mg 3) Acetyl L-carnitine250 mg Propionyl L-carnitine 250 mg 4) Acetyl L-carnitine 200 mgPropionyl L-carnitine 200 mg L-carnitine 200 mg ValeryL L-carnitine 200mg Butyryl L-carnitine 200 mg 5) Acetyl L-carnitine 200 mg PropionylL-carnitine 200 mg L-carnitine 100 mg Coenzyme Q₁₀ 25 mg Vitamin E 5 mgVitamin C 100 mg Linoleic acid 50 mg Linolenic acid 50 mg L-cysteine 50mg N-acetylcysteine 50 mg 6) Acetyl L-carnitine 200 mg PropionylL-carnitine 200 mg L-carnitine 200 mg Coenzyme Q₁₀ 20 mg Resveratrol 1mg Vitamin E 5 mg Vitamin C 100 mg β-carotene 2 mg Lycopene 5 mgRiboflavin 10 mg Pyridoxine 10 mg Pantothenic acid 50 mg

What is meant by pharmacologically acceptable salt of L-carnitine oralkanoyl L-carnitine is any salt of these with an acid that does notgive rise to unwanted toxic or side effects. Such acids are well knownto pharmacologists and to experts in pharmaceutical technology.

Examples of such acids, though not exclusively these, are: chloride;bromide; iodide; aspartate, acid aspartate; citrate, acid citrate;tartrate; phosphate, acid phosphate; fumarate, acid fumarate; glycerolphosphate; glucose phosphate; lactate; maleate, acid maleate; orotate;oxalate, acid oxalate; sulphate, acid sulphate; trichloroacetate;trifluoroacetate and methane sulphonate.

A list of FDA-approved pharmacologically acceptable salts is given inInt. J. of Pharm. 33, 1986, 201-217. This publication is incorporatedherein by reference.

The compositions of the invention can be formulated as tablets,lozenges, pills, capsules, granulates, syrups, vials or drops.

1. A method for providing kidney protection from a kidney dysfunction caused by lithium which comprises administering to an individual in need thereof a therapeutically effective amount of acetyl L-carnitine and propionyl L-carnitine or a pharmacologically acceptable salt thereof, in which the weight-ratio of acetyl L-carnitine to propionyl L-carnitine is 1:1.
 2. A method for providing protection from a kidney dysfunction caused by lithium which comprises administering to an individual in need thereof a combination composition of a therapeutically effective amount of acetyl L-carnitine and propionyl L-carnitine or a pharmacologically acceptable salt thereof, in which the weight-ratio of acetyl L-carnitine to propionyl L-carnitine is 1:1.
 3. A method for providing protection from nephropathy caused by lithium which comprises administering to an individual in need thereof a combination composition of a therapeutically effective amount of acetyl L-carnitine and propionyl L-carnitine or a pharmacologically acceptable salt thereof, in which the weight-ratio of acetyl L-carnitine to propionyl L-carnitine is 1:1.
 4. A method for providing protection from tubular necrosis caused by lithium which comprises administering to an individual in need thereof a therapeutically effective amount of a combination composition of a therapeutically effective amount of acetyl L-carnitine and propionyl L-carnitine or a pharmacologically acceptable salt thereof, in which the weight-ratio of acetyl L-carnitine to propionyl L-carnitine is 1:1.
 5. A method for providing protection from tubular necrosis caused by lithium which comprises administering to an individual in need thereof a therapeutically effective amount of acetyl L-carnitine and propionyl L-carnitine or a pharmacologically acceptable salt thereof, in which the weight-ratio of acetyl L-carnitine to propionyl L-carnitine is 1:1.
 6. A method for providing protection from tubular necrosis caused by lithium which comprises administering to an individual in need thereof 2-5 mg/kg body weight/day of acetyl L-carnitine and 2-5 mg/kg body weight/day of propionyl L-carnitine or an equimolar amount of a pharmacologically acceptable salt thereof, in which the weight-ratio of acetyl L-carnitine to propionyl L-carnitine is 1:1.
 7. A method for providing protection from a kidney dysfunction caused by lithium which comprises administering to an individual in need thereof a combination composition of 2-5 mg/kg body weight/day of acetyl L-carnitine and 2-5 mg/kg body weight/day of propionyl L-carnitine or an equimolar amount of a pharmacologically acceptable salt thereof, in which the weight-ratio of acetyl L-carnitine to propionyl L-carnitine is 1:1.
 8. A method for providing protection from nephropathy caused by lithium which comprises administering to an individual in need thereof a combination composition of 2-5 mg/kg body weight/day of acetyl L-carnitine and 2-5 mg/kg body weight/day of propionyl L-carnitine or an equimolar amount of a pharmacologically acceptable salt thereof, in which the weight-ratio of acetyl L-carnitine to propionyl L-carnitine is 1:1.
 9. A method for providing protection from tubular necrosis caused by lithium which comprises administering to an individual in need thereof a therapeutically effective amount of a combination composition of 2-5 mg/kg body weight/day of acetyl L-carnitine and 2-5 mg/kg body weight/day of propionyl L-carnitine or an equimolar amount of a pharmacologically acceptable salt thereof, in which the weight-ratio of acetyl L-carnitine to propionyl L-carnitine is 1:1. 